Microparticles for drug delivery across mucosa and the blood-brain barrier

ABSTRACT

Pharmaceutical composition comprising micro particles having an average diameter ranging from 40 to 150 nm, consisting of one or more lipids, a drug and, optionally, a steric stabilizer, suitable to the transmucosal passage and to the overcoming of the blood-brain barrier and the blood-cerebrospinal fluid barrier, said micro particles being obtained dispersing in an aqueous medium cooled to 2-4° C. an oil/water or a water/oil/water micro emulsion comprising said constituents.

This application is a 371 of PCT/EP98/07644 filed Nov. 27, 1998.

PRIOR ART

An important problem in the field of the administration and theabsorption of drugs consists of the difficulty of the passage for somedrugs through the intestinal mucosa and of the difficulty of the passagethrough the blood-brain barrier and the blood-cerebrospinal fluidbarrier.

In fact the drugs administered by mouth and destined to pass through theintestinal mucosa find a limitation due to the gastrointestinal pH, tothe residence time and to the solubility.

For example biodegradable drugs such as proteins and peptides, andslightly soluble drugs such as some cytostatics are not suitable forthis kind of administration.

On the other hand drugs as the peptides or the antibiotics such asampicillin and the anti-tumour drugs such as the cyclophosphamide arenot able to pass through the blood-brain barrier.

Several researches on the absorption of the colloidal polymericparticles of polystyrene, polyglycol lactates, polyalkilcyanoacrylatesansacrylates and of polymeric liposomes as carriers of drugs by thegastrointestinal tract after the administration by mouth have beencarried out and the passage of said particles through the intestinalmucosa has been proved.

The results of said researches are described for example by A. T.Florence, The Oral Absorption of Micro- and Nanoparticulates:

Neither Exceptional, Not Unusual, Pharm. Res. 14, 259 (1997) and by H.Chen, V. Torchilin, R. Langer, Polymerized Liposomes as Potential OralVaccine: Stability and Bioavailability, J. Controlled Release. 42, 263(1996).

However said colloidal particles have the drawback of a very lowpassage. Moreover the polymers contain traces of solvents anddegradation products which are not pharmaceutically acceptable.

Finally the polystyrene is not acceptable because it is notbiodegradable.

As far as the preparation of compositions suitable to the passagethrough the blood-brain barrier is concerned, nanoparticles ofpolybutylcyanoacrylate containing drugs such as the gentamicin,administered by intravenous way in rats have been used obtaining verypartial results. More often one resorts to the implantation of thecarriers in the skull in the case of brain cancers (Menei P. et al.,Neurosurgery 39, 117 (1996)).

SUMMARY

Pharmaceutical compositions in the form of microparticles suitable tothe passage through the intestinal mucosa, the blood-brain barrier andthe blood-cerebrospinal fluid barrier have been now found.

Said microparticles have a size ranging from 40 to 150 nm, they areformed by one or more lipids optionally in combination with a stericstabilizer and by a drug.

Said microparticles are prepared dispersing in an aqueous medium at 2-4°C. a hot prepared oil/water or water/oil/water microemulsion comprisingone or more lipids, a surfactant agent, a cosurfactant agent andoptionally a steric stabilizer.

DETAILED DESCRIPTION OF THE INVENTION

The characteristics and the advantages of the pharmaceuticalcompositions in the form of microparticles suitable to the passagethrough the intestinal mucosa, the blood-brain barrier and theblood-cerebrospinal fluid barrier according to the present inventionwill be mostly shown during the following description.

Said microparticles are obtained dispersing in an aqueous medium cooledat 2-4° C. an oil/water or water/oil/water microemulsion preparedaccording to the following description.

The preparation by the oil/water microemulsion is carried out by thefollowing steps:

a) a mixture consisting of one or more lipids, at least a surfactantagent and at least a cosurfactant agent is warmed at a temperature atleast equal to the melting temperature of the lipids;

b) a mixture consisting of a drug dissolved or dispersed in water andoptionally a steric stabilizer, warmed at a temperature at least equalto the temperature of the step a) is added to said mixture of step a),obtaining an oil/water microemulsion;

c) the microemulsion is dispersed in an aqueous medium at 2-4° C.obtaining the microparticles in suspension;

d) the microparticles suspension is washed by an aqueous medium bydiafiltration and freeze-dried.

The preparation by the water/oil/water microemulsion is carried out bythe following steps:

a′) a mixture consisting of one or more lipids, at least a surfactantagent and at least a cosurfactant agent is warmed at a temperature atleast equal to the melting temperature of the lipids;

b′) a mixture consisting of a drug dissolved or dispersed in waterwarmed at a temperature at least equal to the temperature of the stepa′) is added to the mixture of step a′) obtaining a water/oilmicroemulsion;

c′) a mixture consisting of water, at least a surfactant agent and atleast a cosurfactant agent and optionally a steric stabilizer warmed ata temperature at least equal to the temperature of the step a′) is addedto said microemulsion obtaining the water/oil/water microemulsion;

d′) said water/oil/water microemulsion is dispersed in an aqueous mediumat 2-4° C. obtaining the microparticles in suspension;

e′) the microparticles suspension is washed by water by diafiltrationand freeze-dried.

The amount of water used in the steps c) and d′) is ranging from 5 to200 volumes per volume of the respective microemulsion.

The obtained microparticles have an average diameter ranging from 40 to150 nm and preferably from 60 to 100 nm and a polydispersion indexranging from 0.15 to 0.28.

The lipids used in the preparation of the microparticles according tothe present invention are selected from the group consisting of thestearic acid, the palmitic acid, the triglycerides, the diglycerides andthe monoglycerides.

The surfactant agents are selected among soy-bean phosphatidylcholine,dioleyl phosphatidylcholine, dipalmitoyl phosphatidylcholine,hydrogenated soy-bean phosphatidylcholine, phosphatidylethanolamine andphosphatidylserine.

The cosurfactant agents are selected among ethanol, propanol,isopropanol, butanol, sodium taurocholate, sodium glycocholate,propylene glycol, butyric acid and benzoic acid.

The possible steric stabilizer is selected among dipalmitoylphosphatidyl ethanolamine-PEG, PEG-stearate, the esters of the fattyacids from the myristic acid to the docosanoic acid with methyl etherPEG, the diacylphosphatidyl ethanolamines esterified with methyl etherPEG and the polylactates and the polyglycolactates esterified withmethyl ether PEG.

The methyl ether PEG has preferably a molecular weight ranging from 750to 2000.

The washing by diafiltration of the steps d) and e′) has the aim toremove the surfactant agent, the cosurfactant agent and the possibledrug not included in the lipid so that the final composition of themicroparticles results:

lipids, from 80 to 99% by weight,

drug, from 1 to 20% by weight, or:

lipids, from 75 to 98.5% by weight,

steric stabilizer, from 0.5 to 15% by weight,

drug, from 1 to 10% by weight.

The microparticles according to the present invention are successfullyused in the preparation of the compositions suitable to theadministration by mouth for the transmucosal absorption directed towardsthe lymphatic system and for the intravenous administration for theovercoming of the blood-brain barrier and the blood-cerebrospinal fluidbarrier.

The compositions containing the steric stabilizer are particularlysuitable for the intravenous administration.

Forms suitable to the administration by mouth are aqueous dispersions,having a microparticle content ranging from 20 to 200 mg/ml.

Forms suitable to the intravenous administration are aqueous dispersionshaving a microparticle content ranging from 30 to 150 mg/ml.

Drugs particularly suitable for the transmucosal way are cytostaticdrugs used for the therapy of the lymphomas such as methotrexate,hydarubicin, cyclophosphamide, vincristine and vinblastine, antibioticssuch as the gentamicin and peptides such as calcitonin, LHRH andanalogous ones.

Drugs particularly suitable for the passage of the blood-brain barrierand the blood-cerebrospinal fluid barrier are the peptides such as LHRHand analogous ones, enkephalins, antibiotics such as ampicillin andgentamicin and anti-tumour drugs such as the cyclophosphamide andderivatives, carmustine and carboplatinum.

As far as the transmucosal transport is concerned we have the advantagethat the transport by lymphatic way avoids the first passage through theliver, it allows the administration by mouth of drugs, such as lipids,for which such administration is not always possible and it allows thetreatment of the lymphatic system cancers.

As far as the transport through the blood-brain barrier and theblood-cerebrospinal fluid barrier is concerned, the compositionsaccording to the present invention allow the passage of drugs includedin the carriers which normally do not pass or pass in an insufficientamount to obtain a suitable therapeutic effect.

Pharmacological Experimentation

Radio-labelled microparticles have been prepared acting according to theExample 3, reported below, and adding a solution of 131iodoheptadecanoic acid to the warm oil/water microemulsion.

The experimentation has been carried out in male albin rats of a Wistarstrain (Charles River-Italy) having a weight equal to 550-650 g.

Doses equal to 10 mg/kg (about 40 μCi) of an aqueous dispersion ofmicroparticles have been administered at the duodenal level to differentgroups of rats.

Samples of lympha from the thoracic duct and blood from the jugular veinhave been taken at different times.

The data are reported in the Table 1 wherein the radioactivitypercentage is reported with respect to the dose administered per gram oflympha and per gram of blood as a function of the time.

TABLE 1 Average values of radioactivity percentage of the administereddose per gram of lympha (A) or of blood (B) determined in time by gammacounting. Time after A B the administration radioactivity %/Lympha g.radioactivity %/Blood g. 30′ 1 0.09 60′ 1.5 0.08 90′ 3 0.08 120′  9 0.07150   8 0.07 180   7.5 0.07

The same dispersion has been administered by intravenous way to the rats(10 mg/kg) (about 30 μCi). The passage through the blood-brain barrieris pointed out by the presence in the liquor of about 5% of the totalradioactivity after 10 minutes from the administration.

For illustrative aim the following Examples of microparticlespreparations according to the present invention are reported.

EXAMPLE 1

a) A mixture consisting of 180 mg of stearic acid, 92 mg ofphosphatidylcholine, 12 mg of dioleyl phosphatidylcholine, 72 mg ofbutanol and 16 mg of butyric acid is warmed at 70° C.

b) 28 mg of an aqueous solution of LHRH (40 mg/ml) warmed at 70° C., areadded under stirring to the mixture of the step a) obtaining a water/oilmicroemulsion;

c) A mixture consisting of 274.4 mg of water, 16.8 mg of soy-beanphosphatidylcholine, 20.8 mg of butanol, 32.4 mg of sodium taurocholateand 4.0 mg of butyric acid, warmed at 70° C., is added under stirring to52 mg of the water/oil microemulsion of the step b) obtaining awater/oil/water microemulsion.

d) The water/oil/water microemulsion of the step c) is dispersed in aratio equal to 1:10 in water at a temperature equal to 2-3° C. obtainingthe microparticles in an aqueous dispersion.

e) The aqueous dispersion of the step d) is washed three times withwater by diafiltration and subsequently it is freeze-dried.

The following results are obtained:

drug incorporated in the microparticles: 80%;

average diameter of the microparticles.: 96 nm;

polydispersion index: 0.23.

EXAMPLE 2

A mixture consisting of 273.6 mg of water, 16.4 mg ofphosphatidylcholine, 20.0 mg of butanol, 32.0 mg of sodium taurocholate,2.4 mg of butyric acid and 2.8 mg of dipalmitoyl phosphatidylethanolamine-PEG (steric stabilizer), warmed at 70° C., is added understirring to 52.0 mg of the water/oil microemulsion of the step b) of theExample 1.

The preparation is completed according to the steps d) and e) of theExample 1.

The following results are obtained:

drug incorporated in the microparticles: 78%;

average diameter of the microparticles: 100 nm;

polydispersion index: 0.24.

EXAMPLE 3

a) A mixture consisting of 24 mg of stearic acid and 6.0 mg ofmonostearine is warmed at 70° C.;

b) a mixture consisting of 6.0 mg of gentamicin, 56.0 mg of sodiumtaurocholate, 28.0 mg of soy-bean phosphatidylcholine and 280.0 mg ofwater, warmed at 70° C., is added under stirring to the mixture of thestep a) obtaining an oil in water microemulsion.

The microemulsion obtained in the step b) is then treated as describedin the steps d) and e) of the Example 1.

The following results are obtained:

gentamicin incorporated in the microparticles: 80%;

average diameter of the microparticles: 105 nm;

polydispersion index: 0.22.

EXAMPLE 4

a) A mixture consisting of 210.0 mg of stearic acid, 10.0 mg ofmonostearine, 62.0 mg of soy-bean phosphatidylcholine, 80.0 mg ofbutyric acid and 6.0 mg of butanol is warmed at 70° C.

b) 36.0 mg of an aqueous solution of calcitonin (2 mg/ml) warmed at 68°C. are added to the mixture of the step a) obtaining a water/oilmicroemulsion.

c) A mixture consisting of 214.4 mg of water, 16.8 mg of soy-beanphosphatidylcholine, 16.4 g of butyric acid, 8.0 g of butanol and 32.4 gof sodium taurocholate, warmed at 70° C., is added under stirring to52.0 mg of the water/oil microemulsion of the step b), obtaining awater/oil/water microemulsion.

The preparation is then completed working according to the steps d) ande) of the Example 1.

The following results are obtained:

incorporation of the calcitonin: 83%;

average diameter of the microparticles: 93 nm;

polydispersion index: 0.23.

EXAMPLE 5

A mixture consisting of 212.4 mg of water, 16.8 mg of soy-beanphosphatidylcholine, 16.4 mg of butyric acid, 8.0 g of butanol, 32.4 mgof sodium taurocholate and 8.0 mg of stearic acid-PEG (stericstabilizer) warmed at 68° C. is added under stirring to 52.0 mg of thewater/oil microemulsion of the step b) of the Example 4.

The preparation is completed according to the steps d) and e) of theExample 1.

The following results are obtained:

incorporation of the calcitonin: 70%;

average diameter of the microparticles: 102 nm;

polydispersion index: 0.23.

What is claimed is:
 1. Compositions suitable to oral administration fortransmucosal absorption and to intravenous administration for overcomingof the blood-brain barrier and the blood-cerebrospinal fluid barrier,comprising aqueous dispersions of microparticles having an averagediameter ranging from 40 to 150 nm and a polydispersion index rangingfrom 0.15 to 0.28, said microparticles consisting of one or more lipids,a drug and, optionally, a stabilizing agent selected from the groupconsisting of dipalmitoyl phosphatidyl ethanolamine-PEG, PEG-stearate,esters of the fatty acids from myristic acid to docosanoic acid withmethyl ether PEG, diacylphosphatidyl ethanolamines esterified withmethyl ether PEG and polylactates and polyglycolactates esterified withmethyl ether PEG, said microparticles prepared by dispersing in anaqueous medium, cooled at 2 to 4° C., an oil in water or awater/oil/water microemulsion warmed at a temperature near the meltingtemperature of the lipids, washing with water by diafiltration andfreeze-drying.
 2. Compositions as claimed in claim 1, wherein saidmicroparticles are present in said aqueous dispersions in an amountranging from 20 to 200 mg/ml.
 3. Compositions as claimed in claim 1,wherein said microparticles consist of one or more lipids in an amountranging from 80 to 99% by weight and of a drug in an amount ranging from1 to 20% by weight.
 4. Compositions as claimed in claim 1, wherein saidmicroparticles consist of one or more lipids in an amount ranging from75 to 98.5% by weight, of said stabilizing agent in an amount rangingfrom 0.5 to 15% by weight and of a drug in an amount ranging from 1 to10% by weight.
 5. Compositions as claimed in claim 1, wherein saidlipids are selected from the group consisting of stearic acid, palmiticacid, triglycerides, diglycerides and monoglycerides.
 6. Compositions asclaimed in claim 1, wherein said drug is selected from the groupconsisting of methotrexate, hydarubicin, cyclophosphamide, vincristine,vinblastine, gentamicin, calcitonin, LHRH, enkephalins, ampicillin,gentamicin, carmustine and carboplatinum.
 7. Compositions as claimed inclaim 1, wherein said microparticles are obtained by dispersing, in anaqueous medium cooled at 2-4° C., an oil in water or a water/oil/watermicroemulsion warmed at a temperature near the melting temperature ofthe lipids, washing with water by diafiltration and freeze-drying.